SPECIFIC AIMS Abstract Mutations in the NF1 tumor suppressor gene cause neurofibromatosis type 1 (NF1), one of the most common human genetic disorders. NF1 encodes a GTPase activating protein (GAP) for p21ras (Ras) called neurofibromin. Individuals with NF1 have a wide range of malignant and nonmalignant manifestations, including the pathognomonic cutaneous neurofibromas and plexiform neurofibromas. Neurofibromas are composed of Schwann cells, endothelial cells, fibroblasts, degranulating, inflammatory mast cells, and pericytes. Studies in cutaneous, mammary, and pancreatic cancers have emphasized the role of inflammatory cells in altering the microenvironment and facilitating malignant outgrowth. Similarly, utilizing genetically engineered mice that are deficient in the murine homologue of NF1 (Nfl), our collaborator, Dr. Parada (Zhu, Science, 2002), found that haploinsufficiency of Nfl in cells in the tumor microenvironment was required for development of neurofibromas. Based on these prior insights, a major focus of this application will be to functionally define how the interactions between the known Nfl +/- cells in the microenvironment promote neurofibroma formation utilizing human and murine cells. Defining the molecular nature of these interactions in promoting tumorigenesis is critical for identifying drug targets to be used in preclinical trials. Our group previously provided the first genetic, cellular, and biochemical evidence that haploinsufficiency of Nfl alters Ras activity and cell fates in mast cells (Ingram, JEM, 2000, 2001). Mast cells release a diverse number of growth factors and other molecules that collectively promote angiogenesis, the alteration of extracellular matrix and cell growth. There is a marked increase in angiogenesis, and increased local concentrations of a unique profile of growth factors in neurofibromas, which we will test as specific therapeutic targets. Our recent studies focused on the role of haploinsufficiency of Nfl in modulating mechanisms underlying the recruitment of mast cells to the tumor microenvironment (Yang, JCI2003). We now propose to extend these observations to examine the cellular and biochemical mechanisms underlying the role of mast cells in altering Nfl -/- Schwann cell fates, and the function of Nfl +/- fibroblasts, which are important components of the neurofibroma microenvironment. Preliminary studies have identified haploinsufficient phenotypes in Nfl +/- fibroblasts. Studies proposed in this application are designed to further examine the influence of mast cell-derived growth factors and cytokines on the function of Nfl haploinsufficient fibroblasts and the complementary influence of fibroblasts on both mast cell function and tumor progression. Additionally, Dr. Ingram (project 3) has demonstrated previously unrecognized haploinsufficient phenotypes in Nfl +/- endothelial cells and vascular smooth muscle cells, which are cell types that have recently received intense investigation as therapeutic targets. We hypothesize that Nfl+l- fibroblasts, endothelial cells, and vascular smooth muscle cells have in vivo haploinsufficient phenotypes, that are further perturbed by secretory proteins released from Nfl +/- mast cells. We will test these hypotheses with in vivo studies, utilizing genetic intercrosses and pharmacologic agents currently used to treat other cancers and stateof- the-art cellular and tissue imaging. Finally, cellular and biochemical studies will be conducted in primary human fibroblasts and mast cells to verify that the murine model faithfully recapitulates the gains-in-function observed in human NF1 cells.